Neurofibromatosis 1 (NF1) is a common genetic disorder characterized by abnormalities of tissues derived from the neural crest. To define germ-line mutations in the NF1 gene, we studied 20 patients with familial or sporadic cases of NF1 diagnosed clinically and one patient with only café-au-lait spots and no other diagnostic criteria. A protein truncation assay identified abnormal polypeptides synthesized in vitro from five RT-PCR products that represented the entire NF1 coding region. Truncated polypeptides were observed in 14 individuals. The mutations responsible for the generation of abnormal polypeptides were characterized by DNA sequencing. Thirteen previously unpublished mutations were characterized in the 14 individuals. The mutation 2027insC was observed in two unrelated individuals; the other 12 mutations were unique. The sequence changes included seven nonsense and four frameshift mutations that created premature translation termination signals, and two large in-frame deletions that led to the synthesis of truncated polypeptides. One of the mutations was found in the child with a single clinical diagnostic criterion, providing her with a presumptive diagnosis of NF1. Our results confirm that truncating mutations are frequent in both familial and sporadic NF1 cases. The identification of mutations in 14 of 21 individuals studied (67%) suggests that the use of protein truncation assays will rapidly accelerate the rate of identification of NF1 mutations. Because we scanned the entire NF1 coding region in each individual, the distribution of NF1 truncating mutations was discerned for the first time. The mutations were relatively evenly distributed throughout the coding region with no evidence for clustering.

Human fibrinogen has been expressed in Chinese hamster ovary (CHO) cells using a novel two-step procedure which permits efficient synthesis of engineered variant fibrinogens. CHO cells secreting recombinant fibrinogen were grown in roller bottles and maintained in serum-free media for several months. Recombinant protein was purified from media containing 2-4 micrograms/mL fibrinogen using protamine-Sepharose chromatography. Recombinant fibrinogen was identical to plasma fibrinogen when examined on Coomassie-stained SDS gels run under reducing conditions, and on SDS gels when run under nonreducing conditions after partial or complete plasmin degradation, indicating normal chain assembly, disulfide bond formation, and overall protein conformation. Thrombin digestion of purified fibrinogen led to clot formation with release of normal fibrinopeptides, as identified by HPLC. Fibrinopeptide A released from recombinant fibrinogen was partially phosphorylated (22%), similar to the degree of phosphorylation found for human plasma fibrinogen (20-25%), indicating that partial phosphorylation in inherent in fibrinogen synthesis.

During cleavage of fibrinogen by thrombin, fibrinopeptide A (FpA) release precedes fibrinopeptide B (FpB) release. To examine the basis for this ordered release, we synthesized A␤ fibrinogen, replacing FpB with a fibrinopeptide A-like peptide, FpA (G14V). Analyses of fibrinopeptide release from A␤ fibrinogen showed that FpA release and FpA release were similar; the release of either peptide followed simple first-order kinetics. Specificity constants for FpA and FpA were similar, demonstrating that these peptides are equally competitive substrates for thrombin. In the presence of Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, the rate of FpB release from normal fibrinogen was reduced 3-fold, consistent with previous data; in contrast, the rate of FpA release from A␤ fibrinogen was unaffected. Thus, with A␤ fibrinogen, fibrinopeptide release from the ␤ chain is similar to fibrinopeptide release from the ␣ chain. We conclude that the ordered release of fibrinopeptides is dictated by the specificity of thrombin for its substrates. We analyzed polymerization, following changes in turbidity, and found that polymerization of A␤ fibrinogen was similar to that of normal fibrinogen. We analyzed clot structure by scanning electron microscopy and found that clots from A␤ fibrinogen were similar to clots from normal fibrinogen. We conclude that premature release of the fibrinopeptide from the N terminus of the ␤ chain does not affect polymerization of fibrinogen.Fibrinogen is a 340-kDa plasma protein that is involved in the final phase of the coagulation cascade. Fibrinogen consists of two pairs of three polypeptide chains (A␣, B␤, and ␥) that fold to produce a trinodular protein with two distal (D) nodules connected to a central nodule (E) by coiled-coil regions. The central nodule of the molecule consists of the N termini of all six polypeptide chains, and the D nodules consist predominantly of the C termini of the ␤ and ␥ chains, each folded into a globular domain. To initiate polymerization, the serine protease thrombin cleaves four specific Arg-Gly bonds at the N termini of both the A␣ and B␤ chains, releasing fibrinopeptides A (FpA) 1 and B (FpB), respectively. The release of FpA, a 16-residue peptide, exposes the "A" site, which noncovalently interacts with the "a" site in the ␥ chain of the D nodule of another molecule. This A:a interaction results in the linear arrangement of half-staggered, double-stranded protofibrils (1). The release of FpB, a 14-residue peptide, exposes the "B" site (2-4), which presumably interacts with a "b" site in the ␤ chain of the D nodule of another molecule (5). This B:b interaction is thought to be responsible for lateral aggregation of protofibrils to form fibers (2) and to be analogous to the A:a interaction; however, the mechanism of this interaction is not yet well understood. The final product of this polymerization is a complex, branching network of fibers.The interactions of thrombin with fibrinogen have been extensively studied (Refs. 6 and 7; for a review, see Ref. 8)....

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SummaryBinding of the synthetic peptide AAKDSDWPEASDEDWNYKAPSGAR, a fibrinogen α27–50 analog, to thrombin was studied by inhibition assays and affinity chromatography. Peptide α27–50 corresponds to a segment of human fibrinogen downstream from the thrombin cleavage site, with cysteine residues at positions 28, 36, 45 and 49 replaced by alanine. The peptide inhibited clotting of fibrinogen with an inhibition constant of 190–400 μM. Cleavage of fibrinopeptides A and B was inhibited by the peptide and the peptide was competitive with fibrinogen for thrombin. Inhibition of the small substrate tosyl-Gly-Pro-Arg-p-nitroaniline was not observed indicating that the peptide did not block the active site of the enzyme. Peptide α27–50 that was covalently linked to Sepharose bound active siteinhibited thrombin at low ionic strength and was eluted at higher salt concentration. The peptide was not cleaved on overnight exposure to thrombin as determined by reverse phase HPLC. In summary, the peptide bound to, but was not a substrate for thrombin. These results suggest that this region of fibrinogen contributes to binding of thrombin.